Identification of a bioactive 51-membered macrolide complex by activation of a silent polyketide synthase in Streptomyces ambofaciens

Abstract

There is a constant need for new and improved drugs to combat infectious diseases, cancer, and other major life-threatening conditions. The recent development of genomics-guided approaches for novel natural product discovery has stimulated renewed interest in the search for natural product-based drugs. Genome sequence analysis of Streptomyces ambofaciens ATCC23877 has revealed numerous secondary metabolite biosynthetic gene clusters, including a giant type I modular polyketide synthase (PKS) gene cluster, which is composed of 25 genes (nine of which encode PKSs) and spans almost 150 kb, making it one of the largest polyketide biosynthetic gene clusters described to date. The metabolic product(s) of this gene cluster are unknown, and transcriptional analyses showed that it is not expressed under laboratory growth conditions. The constitutive expression of a regulatory gene within the cluster, encoding a protein that is similar to Large ATP binding of the LuxR (LAL) family proteins, triggered the expression of the biosynthetic genes. This led to the identification of four 51-membered glycosylated macrolides, named stambomycins A-D as metabolic products of the gene cluster. The structures of these compounds imply several interesting biosynthetic features, including incorporation of unusual extender units into the polyketide chain and in trans hydroxylation of the growing polyketide chain to provide the hydroxyl group for macrolide formation. Interestingly, the stambomycins possess promising antiproliferative activity against human cancer cell lines. Database searches identify genes encoding LAL regulators within numerous cryptic biosynthetic gene clusters in actinomycete genomes, suggesting that constitutive expression of such pathway-specific activators represents a powerful approach for novel bioactive natural product discovery.

Fig. 1.

Stambomycin biosynthetic gene cluster. (A) Organization of the stambomycin biosynthetic gene cluster. Limits of the cluster have been assigned using sequence comparisons. (B) Module and domain organization of the PKS encoded by the cluster, assigned using the SEARCHPKS program (13). The ketoreductase domain in module 24 is predicted to be nonfunctional because the tyrosine and asparagine residues of the catalytic triad are absent (42). ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; ER^R, enoyl reductase predicted to generate a 2R acyl thioester intermediate; ER^S, enoyl reductase predicted to generate a 2S acyl thioester intermediate; KR*, ketoreductase predicted to generate a 2R, 3R-acyl thioester intermediate; KR⁺, ketoreductase predicted to generate a 2R, 3S-acyl thioester intermediate; KR^#, ketoreductase predicted to generate a 2S, 3S-acyl thioester intermediate; KR^{^}, ketoreductase predicted to generate a 2S, 3R-acyl thioester intermediate; KS, β-ketoacyl synthase; KS^Q, β-ketoacyl synthase in which the active site cysteine residue is replaced by glutamine; TE, thioesterase. (C) Predicted structure of the fully assembled polyketide chain attached to the ACP domain in the last module of the PKS. The structure of the side chain at C-26 could not be predicted because the nature of the substrate of the AT domain in module 12 could not be inferred from sequence comparisons. Atoms in the polyketide chain are colored according to the precursors from which they derive: blue, malonyl-CoA; red, methylmalonyl-CoA; purple, malonyl-CoA derivative of unknown structure.

Fig. 2.

Induction of PKS gene expression. Transcriptional analysis by RT-PCR of the control ATCC/pIB139 and the mutant strain ATCC/OE484, grown in MP5 medium. Expression of four biosynthetic genes (samR0467, samR0465, samR0477, samR0474), together with expression of the regulatory gene samR0484, were analyzed by RT-PCR using 4 μg total RNA. The constitutively expressed hrdB gene, coding for the major sigma factor, was used as positive control. Experiments carried out on three separate occasions gave the same results. T1, exponential phase; T2, transition phase; T3, stationary phase.

Fig. 3.

Detection of the stambomycins. Base peak chromatograms from LC-ESI-TOF-MS analyses of methanolic mycelial extracts of ATCC/pIB139 (Top), ATCC/OE484 (Middle), and ATCC/OE484/Δ467 (Bottom) strains. Peaks corresponding to stambomycins A/B and C/D in the ATCC/OE484 strain are labeled.

Fig. 4.

Structure elucidation of the stambomycins. Structures of stambomycins A–D proposed on the basis of HRMS and NMR spectroscopic analyses. Key COSY, HMBC, and NOESY correlations for structural elements that were not predicted by sequence analysis of the biosynthetic enzymes are indicated by bold lines, single-headed arrows, and double-headed arrows, respectively. ³J coupling constants observed in ¹H NMR spectra for H-12/H-13 and H-49/H-50 are indicated.